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1.
Science ; 367(6482): 1151-1156, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32139547

RESUMO

The regulation of messenger RNA levels in mammalian cells can be achieved by the modulation of synthesis and degradation rates. Metabolic RNA-labeling experiments in bulk have quantified these rates using relatively homogeneous cell populations. However, to determine these rates during complex dynamical processes, for instance during cellular differentiation, single-cell resolution is required. Therefore, we developed a method that simultaneously quantifies metabolically labeled and preexisting unlabeled transcripts in thousands of individual cells. We determined synthesis and degradation rates during the cell cycle and during differentiation of intestinal stem cells, revealing major regulatory strategies. These strategies have distinct consequences for controlling the dynamic range and precision of gene expression. These findings advance our understanding of how individual cells in heterogeneous populations shape their gene expression dynamics.


Assuntos
Estabilidade de RNA , RNA Mensageiro/metabolismo , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Transcrição Genética , Animais , Humanos , Indicadores e Reagentes/química , Células K562 , Camundongos , Uridina/análogos & derivados
2.
PLoS One ; 15(3): e0223030, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32119673

RESUMO

Numerous studies show that various genes in all kinds of organisms are transcribed discontinuously, i.e. in short bursts or pulses with periods of inactivity between them. But it remains unclear whether ribosomal DNA (rDNA), represented by multiple copies in every cell, is also expressed in such manner. In this work, we synchronized the pol I activity in the populations of tumour derived as well as normal human cells by cold block and release. Our experiments with 5-fluorouridine (FU) and BrUTP confirmed that the nucleolar transcription can be efficiently and reversibly arrested at +4°C. Then using special software for analysis of the microscopic images, we measured the intensity of transcription signal (incorporated FU) in the nucleoli at different time points after the release. We found that the ribosomal genes in the human cells are transcribed discontinuously with periods ranging from 45 min to 75 min. Our data indicate that the dynamics of rDNA transcription follows the undulating pattern, in which the bursts are alternated by periods of rare transcription events.


Assuntos
DNA Ribossômico/genética , Ribossomos/genética , Transcrição Genética , Idoso , Cadáver , Nucléolo Celular/genética , Temperatura Baixa , Células Epiteliais/metabolismo , Células HeLa , Humanos , Cinética , Limbo da Córnea/citologia , Pessoa de Meia-Idade , RNA Ribossômico/genética , Software , Transfecção , Uridina/análogos & derivados , Uridina/imunologia , Uridina/metabolismo , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/imunologia , Uridina Trifosfato/metabolismo
3.
Molecules ; 25(1)2019 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-31861655

RESUMO

Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure-activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 2'-hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates.


Assuntos
Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Nucleosídeos/síntese química , Transferases/antagonistas & inibidores , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/enzimologia , Proteínas de Bactérias/química , Modelos Moleculares , Estrutura Molecular , Nucleosídeos/química , Nucleosídeos/farmacologia , Relação Estrutura-Atividade , Timidina/química , Transferases/química , Uridina/análogos & derivados , Uridina/química
4.
Nucleic Acids Res ; 47(19): 10296-10312, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31495891

RESUMO

Oxazinomycin is a C-nucleoside antibiotic that is produced by Streptomyces hygroscopicus and closely resembles uridine. Here, we show that the oxazinomycin triphosphate is a good substrate for bacterial and eukaryotic RNA polymerases (RNAPs) and that a single incorporated oxazinomycin is rapidly extended by the next nucleotide. However, the incorporation of several successive oxazinomycins or a single oxazinomycin in a certain sequence context arrested a fraction of the transcribing RNAP. The addition of Gre RNA cleavage factors eliminated the transcriptional arrest at a single oxazinomycin and shortened the nascent RNAs arrested at the polythymidine sequences suggesting that the transcriptional arrest was caused by backtracking of RNAP along the DNA template. We further demonstrate that the ubiquitous C-nucleoside pseudouridine is also a good substrate for RNA polymerases in a triphosphorylated form but does not inhibit transcription of the polythymidine sequences. Our results collectively suggest that oxazinomycin functions as a Trojan horse substrate and its inhibitory effect is attributable to the oxygen atom in the position corresponding to carbon five of the uracil ring.


Assuntos
RNA Polimerases Dirigidas por DNA/química , RNA/química , Transcrição Genética/efeitos dos fármacos , Uridina/análogos & derivados , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Oxigênio/química , Pseudomonas/química , RNA/genética , Clivagem do RNA/efeitos dos fármacos , Streptomyces/química , Especificidade por Substrato , Timidina/química , Timidina/genética , Transcrição Genética/genética , Fatores de Elongação da Transcrição/genética , Uracila/química , Uridina/síntese química , Uridina/química , Uridina/farmacologia
5.
Hum Cell ; 32(4): 477-486, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31428943

RESUMO

MicroRNAs (miRNAs) are defined as small, non-coding RNAs that act as post-transcriptional regulators of gene expression. Dysfunction of miRNAs was involved in the initiation and progression of nasopharyngeal carcinoma (NPC). Here, we found that miR-543 was markedly overexpressed in NPC tissues and cell lines. Overexpression of miR-543 promoted the proliferation, cell cycle progression and invasion of NPC cells. Down-regulation of miR-543 inhibited the proliferation and induced apoptosis of NPC cells. Bioinformatics analysis suggested the junctional adhesion molecule A (JAM-A) as a potential target of miR-543. Furthermore, molecular study showed that the miR-543 bound the 3'-untranslated region (UTR) of JAM-A and decreased the expression of JAM-A in NPC cells. The expression of JAM-A in NPC tissues was decreased and negatively correlated with that of miR-543. Overexpression of JAM-A attenuated miR-543-induced proliferation of NPC cells. Collectively, these evidence indicated the important roles of miR-543/JAM-A signaling in the progression of NPC, highlighting the potential of miR-543 as a target in the treatment of NPC.


Assuntos
Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/genética , MicroRNAs/fisiologia , Carcinoma Nasofaríngeo/genética , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/patologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Expressão Gênica/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Terapia de Alvo Molecular , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/terapia , Invasividade Neoplásica/genética , Ácidos Fosfatídicos/genética , Transdução de Sinais , Uridina/análogos & derivados , Uridina/genética
6.
PLoS Genet ; 15(8): e1008117, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31465447

RESUMO

The Elongator complex promotes formation of 5-methoxycarbonylmethyl (mcm5) and 5-carbamoylmethyl (ncm5) side-chains on uridines at the wobble position of cytosolic eukaryotic tRNAs. In all eukaryotic organisms tested to date, the inactivation of Elongator not only leads to the lack of mcm5/ncm5 groups in tRNAs, but also a wide variety of additional phenotypes. Although the phenotypes are most likely caused by a translational defect induced by reduced functionality of the hypomodified tRNAs, the mechanism(s) underlying individual phenotypes are poorly understood. In this study, we show that the genetic background modulates the phenotypes induced by the lack of mcm5/ncm5 groups in Saccharomyces cerevisiae. We show that the stress-induced growth defects of Elongator mutants are stronger in the W303 than in the closely related S288C genetic background and that the phenotypic differences are caused by the known polymorphism at the locus for the mRNA binding protein Ssd1. Moreover, the mutant ssd1 allele found in W303 cells is required for the reported histone H3 acetylation and telomeric gene silencing defects of Elongator mutants. The difference at the SSD1 locus also partially explains why the simultaneous lack of mcm5 and 2-thio groups at wobble uridines is lethal in the W303 but not in the S288C background. Collectively, our results demonstrate that the SSD1 locus modulates phenotypes induced by the lack of Elongator-dependent tRNA modifications.


Assuntos
Fatores de Alongamento de Peptídeos/genética , RNA de Transferência/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Expressão Gênica/genética , Genótipo , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Fenótipo , Processamento Pós-Transcricional do RNA/genética , RNA de Transferência/genética , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Uridina/análogos & derivados , Uridina/química
7.
Pestic Biochem Physiol ; 158: 112-120, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31378345

RESUMO

Cytochrome P450s (P450s) confer resistance against herbicides, and this is increasingly becoming a concern for weed control. As a widespread Gramineae weed in paddy fields, Echinocloa glabrescens has become resistant to the acetolactate synthase (ALS)-inhibiting triazolopyrimidine herbicide penoxsulam. In this study, we found that the GR50 of the resistant population (SHQP-R) decreased substantially from 25.6 to 5.0 and 6.2 g a.i. ha-1 after treatment with the P450 inhibitors piperonyl butoxide (PBO) and malathion, respectively. However, P450 inhibitors almost had no effects on the susceptibility of the sensitive population (JYJD-S) to penoxsulam. To investigate the mechanisms of metabolic resistance, transcriptome sequencing analysis was performed to find candidate genes that may confer resistance to penoxsulam in E. glabrescens. A total of 233 P450 differentially expressed genes (DEGs) were identified by transcriptome sequencing. We found that the metabolic process and metabolic pathways were the most highly enriched in DEGs. Further, twenty-seven candidate P450 DEGs were selected for qPCR validation analyses. After penoxsulam treatment, the relative expression levels were significantly higher in SHQP-R than in JYJD-S. Among these, the relative expression of twenty-three P450 DEGs (eighteen from the CYP72A-71C-74A-96A-734A subfamily; five from CYP81E1-94C1-94B3-714C1-714C2) were upregulated and four P450 DEGs (from CYP724B1-711A1-707A7-97B2) were downregulated. Changes in the expression of these candidate P450 genes in E. glabrescens were in response to penoxsulam, which provides preliminary evidence for the role of P450s in herbicide metabolism in E. glabrescens. However, further functional studies on metabolic resistance to penoxsulam in a resistant E. glabrescens population are required.


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Echinochloa/efeitos dos fármacos , Echinochloa/metabolismo , Perfilação da Expressão Gênica/métodos , Sulfonamidas/farmacologia , Uridina/análogos & derivados , Sistema Enzimático do Citocromo P-450/genética , Echinochloa/genética , Resistência a Herbicidas/genética , Malation/farmacologia , Butóxido de Piperonila/farmacologia , Uridina/farmacologia
8.
J Agric Food Chem ; 67(29): 8085-8095, 2019 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-31265279

RESUMO

Herbicide resistance identification is essential for effective chemical weed control. In this study, we quantified the differences in growth response between penoxsulam resistant (R) and sensitive (S) Echinochloa crus-galli populations, explored the changes in ALS, and performed genetic analyses to identify metabolic genes that are up-regulated by the application of penoxsulam and other common herbicides. The R population showed a 26.0-fold higher resistance to penoxsulam and varied resistance to most tested herbicides with indices ranging from 4.9 to 145.9. A Trp-574-Arg amino acid mutation in ALS and low penoxsulam ALS sensitivity were the main mechanisms underlying herbicide resistance. The penoxsulam resistance can be significantly reversed by two P450s inhibitors and one GST inhibitor. By RNA-Seq, thirty-six highly expressed contigs were selected, and 30 of them were up-regulated in the R population treated by penoxsulam. Many of these genes were significantly expressed when treated with pyroxsulam, metamifop, and quinclorac. These upregulated genes appear to be complementary for plant resistance to penoxsulam and other common herbicides.


Assuntos
Echinochloa/efeitos dos fármacos , Resistência a Herbicidas , Herbicidas/farmacologia , Sulfonamidas/farmacologia , Uridina/análogos & derivados , Acetolactato Sintase/genética , Acetolactato Sintase/metabolismo , Echinochloa/genética , Echinochloa/crescimento & desenvolvimento , Echinochloa/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Uridina/farmacologia
9.
J Antibiot (Tokyo) ; 72(10): 769-774, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31341273

RESUMO

A novel sansanmycin analogue, sansanmycin Q (1), was identified by genome mining from the fermentation broth of Streptomyces sp. SS (CPCC 200442). In comparison with other sansanmycin compounds, sansanmycin Q has an extra glycine residue at the N-terminus of the pseudopeptide backbone. The additional glycine was proved to be assembled to sansanmycin A by SsaB, a tRNA-dependent aminoacyltransferase, based on the results of rescrutiny of sansanmycin biosynthetic gene cluster, and then overexpression and knockout of ssaB in the wild-type strain. The structure of sansanmycin Q was assigned by interpretation of NMR and mass spectral data. The results of the bioassay disclosed that sansanmycin Q exhibited more potency against Mycobacterium tuberculosis H37Rv and a rifampicin- and isoniazid-resistant strain than sansanmycin A.


Assuntos
Antituberculosos/metabolismo , Antituberculosos/farmacologia , Vias Biossintéticas/genética , Família Multigênica , Oligopeptídeos/biossíntese , Oligopeptídeos/farmacologia , Streptomyces/metabolismo , Uridina/análogos & derivados , Antituberculosos/química , Biologia Computacional , Fermentação , Genoma Bacteriano , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Estrutura Molecular , Mycobacterium tuberculosis/efeitos dos fármacos , Oligopeptídeos/química , Streptomyces/crescimento & desenvolvimento , Uridina/biossíntese , Uridina/química , Uridina/farmacologia
10.
Adv Mater ; 31(32): e1902672, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31206855

RESUMO

Cancer theranostics holds potential promise for precision medicine; however, most existing theranostic nanoagents are simply developed by doping both therapeutic agents and imaging agent into one particle entity, and thus have an "always-on" pharmaceutical effect and imaging signals regardless of their in vivo location. Herein, the development of an organic afterglow protheranostic nanoassembly (APtN) that specifically activates both the pharmaceutical effect and diagnostic signals in response to a tumor-associated chemical mediator (hydrogen peroxide, H2 O2 ) is reported. APtN comprises an amphiphilic macromolecule and a near-infrared (NIR) dye acting as the H2 O2 -responsive afterglow prodrug and the afterglow initiator, respectively. Such a molecular architecture allows APtN to passively target tumors in living mice, specifically release the anticancer drug in the tumor, and spontaneously generate the uncaged afterglow substrate. Upon NIR light preirradiation, the afterglow initiator generates singlet oxygen to react and subsequently transform the uncaged afterglow substrate into an active self-luminescent form. Thus, the intensity of generated afterglow luminescence is correlated with the drug release status, permitting real-time in vivo monitoring of prodrug activation. This study proposes a background-free design strategy toward activatable cancer theranostics.


Assuntos
Antineoplásicos/síntese química , Substâncias Luminescentes/química , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Pró-Fármacos/síntese química , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Dimerização , Sistemas de Liberação de Medicamentos , Floxuridina/química , Peróxido de Hidrogênio/metabolismo , Raios Infravermelhos , Camundongos , Polietilenoglicóis/química , Pró-Fármacos/farmacologia , Nanomedicina Teranóstica , Distribuição Tecidual , Microambiente Tumoral , Uridina/análogos & derivados , Uridina/química
11.
Eur J Med Chem ; 171: 462-474, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30933853

RESUMO

The present status of antibiotic resistant requires an urgent invention of novel agents that act on clinically unexplored antibacterial targets. The enzyme MraY (phospho-MurNAc-pentapeptide translocase), essential for bacterial cell wall synthesis, fulfils this criterion as it has not been explored as a target in a clinical context. Specifically, the enzyme is involved in the lipid-linked cycle of peptidoglycan biosynthesis and is reportedly targeted by naturally-occurring nucleoside antibiotics. The antimicrobial 'caprazamycin' class of nucleoside antibiotics targets Mycobacterium tuberculosis and clinically relevant Gram-negative bacteria such as Pseudomonas aeruginosa besides various drug resistant strains and is therefore an eligible starting point for the development of novel agents. In this review, we aim to summarise the structure-activity relationships of the natural, semi-synthetic as well as synthetic analogues of nucleoside antibiotic caprazamycins. This review highlights caprazamycins as promising lead structures for development of potent and selective antimicrobial agents that target MraY, the bacterial enzyme involved in the first membrane-dependent step in bacterial peptidoglycan assembly.


Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Produtos Biológicos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Transferases/antagonistas & inibidores , Uridina/análogos & derivados , Antibacterianos/química , Azepinas/química , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade , Transferases/metabolismo , Uridina/química , Uridina/farmacologia
12.
Bull Environ Contam Toxicol ; 102(6): 854-860, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30989281

RESUMO

Photodegradation is an important non-biodegradation process of pesticide degradation in aquatic environments. In this study, the effect of different forms of nitrogen on the photodegradation kinetics of penoxsulam was investigated. The photodegradation of penoxsulam was accelerated by NO3- and NO2- but was not affected by NH4+. Ultra-high-performance liquid chromatography coupled with time-of-flight mass spectrometry was used to separate and identify the transformation products (TPs)converted by photodegradation of penoxsulam in an aqueous solution under UV-Vis (290-800 nm) irradiation. Seven major transformation products were identified based on mass spectral data. The structure was determined by elemental composition calculations, comparison of structural analogs, and existing literature. The main pathways of photodegradation were found to be sulfonamide bond cleavage, rearrangement, triazole ring cleavage, and hydroxylation. These findings are critical to elucidate the environmental fate of penoxsulam in aquatic ecosystems and provide a basis for further environmental risk assessment.


Assuntos
Herbicidas/química , Fotólise , Sulfonamidas/química , Uridina/análogos & derivados , Poluentes Químicos da Água/química , Amônia/química , Cromatografia Líquida de Alta Pressão , Herbicidas/análise , Cinética , Espectrometria de Massas/métodos , Óxido Nítrico/química , Medição de Risco , Sulfonamidas/análise , Raios Ultravioleta , Uridina/análise , Uridina/química , Água/química , Poluentes Químicos da Água/análise
13.
Int J Hematol ; 110(2): 161-169, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31020568

RESUMO

Hypomethylating agents (HMAs), azacitidine and decitabine, are standards of care in higher-risk myelodysplastic syndromes and in acute myeloid leukemia patients ineligible for intensive therapy. Over the last 10 years, research efforts have sought to better understand their mechanism of action, both at the molecular and cellular level. These efforts have yet to robustly identify biomarkers for these agents. The clinical activity of HMAs in myeloid neoplasms has been firmly established now but still remains of limited magnitude. Besides optimized use at different stages of the disease, most of the expected clinical progress with HMAs will come from the development of second-generation compounds orally available and/or with improved pharmacokinetics, and from the search, so far mostly empirical, of HMA-based synergistic drug combinations.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Azacitidina/uso terapêutico , Metilação de DNA/efeitos dos fármacos , Decitabina/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Síndromes Mielodisplásicas/tratamento farmacológico , Antimetabólitos Antineoplásicos/administração & dosagem , Antimetabólitos Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/administração & dosagem , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Ensaios Clínicos como Assunto , Decitabina/química , Decitabina/farmacologia , Esquema de Medicação , Combinação de Medicamentos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mielomonocítica Crônica/tratamento farmacológico , Leucemia Mielomonocítica Crônica/genética , Síndromes Mielodisplásicas/genética , Uridina/administração & dosagem , Uridina/análogos & derivados , Uridina/farmacologia , Uridina/uso terapêutico
15.
J Plant Res ; 132(3): 395-403, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30847615

RESUMO

The nucleolus, where components of the ribosome are constructed, is known to play an important role in various stress responses in animals. However, little is known about the role of the plant nucleolus under environmental stresses such as heat and chilling stress. In this study, we analyzed nucleolus morphology by determining the distribution of newly synthesized rRNAs with an analog of uridine, 5-ethynyl uridine (EU). When EU was incorporated into the root of the Arabidopsis thaliana, EU signals were strongly localized in the nucleolus. The results of the short-term incorporation of EU implied that there is no compartmentation among the processes of transcription, processing, and construction of rRNAs. Nevertheless, under heat and chilling stress, EU was not incorporated into the center of the nucleolus. Morphological analyses using whole rRNA staining and differential interference contrast observations revealed speckled and round structures in the center of the nucleolus under heat and chilling stress, respectively.


Assuntos
Nucléolo Celular/fisiologia , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Arabidopsis/ultraestrutura , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Resposta ao Choque Frio , Resposta ao Choque Térmico , Uridina/análogos & derivados , Uridina/metabolismo
16.
Molecules ; 24(6)2019 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-30901934

RESUMO

Tick-borne encephalitis virus (TBEV) is a causative agent of tick-borne encephalitis (TBE), one of the most important human infections involving the central nervous system. Although effective vaccines are available on the market, they are recommended only in endemic areas. Despite many attempts, there are still no specific antiviral therapies for TBEV treatment. Previously, we synthesized a series of uridine derivatives of 2-deoxy sugars and proved that some compounds show antiviral activity against viruses from the Flaviviridae and Orthomyxoviridae families targeting the late steps of the N-glycosylation process, affecting the maturation of viral proteins. In this study, we evaluated a series of uridine derivatives of 2-deoxy sugars for their antiviral properties against two strains of the tick-borne encephalitis virus; the highly virulent TBEV strain Hypr and the less virulent strain Neudoerfl. Four compounds (2, 4, 10, and 11) showed significant anti-TBEV activity with IC50 values ranging from 1.4 to 10.2 µM and low cytotoxicity. The obtained results indicate that glycosylation inhibitors, which may interact with glycosylated membrane TBEV E and prM proteins, might be promising candidates for future antiviral therapies against TBEV.


Assuntos
Antivirais/farmacologia , Desoxiaçúcares/farmacologia , Vírus da Encefalite Transmitidos por Carrapatos/efeitos dos fármacos , Uridina/farmacologia , Antivirais/química , Linhagem Celular Tumoral , Células Cultivadas , Desoxiaçúcares/química , Relação Dose-Resposta a Droga , Vírus da Encefalite Transmitidos por Carrapatos/fisiologia , Humanos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Biossíntese de Proteínas/efeitos dos fármacos , Uridina/análogos & derivados , Uridina/química , Ensaio de Placa Viral
17.
Lancet Haematol ; 6(4): e194-e203, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30926081

RESUMO

BACKGROUND: Decitabine, a DNA methyltransferase 1 inhibitor or DNA hypomethylating compound, is not readily orally bioavailable because of rapid clearance by cytidine deaminase (CDA) in the gut and liver. This dose-escalation study, guided by pharmacokinetic and pharmacodynamic observations, evaluated whether simultaneous oral administration with the novel CDA inhibitor cedazuridine increases decitabine bioavailability for the treatment of myelodysplastic syndromes. METHODS: In this phase 1 study, we enrolled patients aged 18 years or older with myelodysplastic syndromes or chronic myelomonocytic leukaemia. Eligible patients were assigned to cohorts to receive escalating oral doses of decitabine and cedazuridine. The starting dose was decitabine 20 mg and cedazuridine 40 mg. Treatment cycles lasted 28 days, with 5 days of drug administration. In cycle 1, each patient received a cohort-defined dose of oral decitabine on day -3, a 1-h intravenous infusion of decitabine 20 mg/m2 on day 1, and cohort-defined doses of oral decitabine plus cedazuridine on days 2-5. In cycles 2 and beyond, the oral decitabine and cedazuridine were given on days 1-5. The dose of cedazuridine was escalated first and decitabine was escalated once CDA inhibition by cedazuridine approached the maximum effect. The drug dose was escalated if mean decitabine area under the curve (AUC) of the oral drug was less than 90% of that for intravenous decitabine in the cohort and if no dose-limiting toxicity was observed. Dose-limiting toxicity was defined as a grade 3 or greater non-haematologic toxicity or grade 4 haematologic toxicity lasting more than 14 days and unrelated to the underlying disease. Once the decitabine AUC target range set as the primary endpoint, and established with intravenous decitabine, was reached at a dose deemed to be safe, the cohort that most closely approximated intravenous decitabine exposure was expanded to 18 evaluable patients. The primary objectives were to assess the safety of decitabine plus cedazuridine, and to determine the dose of each drug needed to achieve a mean AUC for decitabine exposure similar to that for intravenous decitabine exposure. This study is registered with ClinicalTrials.gov, number NCT02103478. FINDINGS: Between Oct 28, 2014, and Nov 13, 2015, we enrolled 44 eligible patients (of 75 screened) with previously treated or newly diagnosed myelodysplastic syndromes or chronic myelomonocytic leukaemia; 43 of the enrolled patients were evaluable. Participants were treated in five cohorts: cohorts 1-4 included six evaluable patients each; cohort 5 included 19 patients in a 13-patient expansion. Dose-dependent increases in decitabine AUC and peak plasma concentration occurred with each cohort dose escalation. There was no evident increase in toxicity compared with that reported for intravenous decitabine. Decitabine 30 mg and 40 mg plus cedazuridine 100 mg produced mean day-5 decitabine AUCs (146 ng × h/mL for decitabine 30 mg, and 221 ng × h/mL for decitabine 40 mg) closest to the mean intravenous-decitabine AUC (164 ng × h/mL). The most common grade 3 or more adverse events were thrombocytopenia (18 [41%] of 44 patients), neutropenia (13 [30%]), anaemia (11 [25%]), leukopenia (seven [16%]), febrile neutropenia (seven [16%]), and pneumonia (seven [16%]). Four (9%) patients died because of adverse events, none of which was considered drug related, and three (7%) patients died more than 30 days after discontinuing treatment because of progressive disease (two [5%]) and respiratory failure (one [2%]). INTERPRETATION: Oral decitabine plus cedazuridine emulated the pharmacokinetics of intravenous decitabine, with a similar safety profile and dose-dependent demethylation. Clinical responses were similar to intravenous decitabine treatment for 5 days. Further study of decitabine plus cedazuridine as an alternative to parenteral therapy or in combination with other new oral agents for myeloid disorders is warranted. FUNDING: Astex Pharmaceuticals, Inc.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Decitabina/uso terapêutico , Síndromes Mielodisplásicas/tratamento farmacológico , Uridina/análogos & derivados , Administração Oral , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Decitabina/administração & dosagem , Decitabina/efeitos adversos , Relação Dose-Resposta a Droga , Interações Medicamentosas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Segurança , Resultado do Tratamento , Uridina/administração & dosagem , Uridina/efeitos adversos , Uridina/uso terapêutico
18.
PLoS One ; 14(2): e0212458, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30817767

RESUMO

BACKGROUND AND AIMS: Inborn errors of purine and pyrimidine metabolism are a diverse group of disorders with possible serious or life-threatening symptoms. They may be associated with neurological symptoms, renal stone disease or immunodeficiency. However, the clinical presentation can be nonspecific and mild so that a number of cases may be missed. Previously published assays lacked detection of certain diagnostically important biomarkers, including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine, necessitating the use of separate assays for their detection. Moreover, the limited sensitivity for some analytes in earlier assays may have hampered the reliable detection of mild cases. Therefore, we aimed to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that allows the simultaneous and sensitive detection of an extended range of purine and pyrimidine biomarkers in urine. METHODS: The assay was developed and validated using LC-MS/MS and clinically tested by analyzing ERNDIM Diagnostic Proficiency Testing (DPT) samples and further specimens from patients with various purine and pyrimidine disorders. RESULTS: Reliable determination of 27 analytes including SAICAr, AICAr, beta-ureidoisobutyric acid, 2,8-dihydroxyadenine and orotidine was achieved in urine following a simple sample preparation. The method clearly distinguished pathological and normal samples and differentiated between purine and pyrimidine defects in all clinical specimens. CONCLUSIONS: A LC-MS/MS assay allowing the simultaneous, sensitive and reliable diagnosis of an extended range of purine and pyrimidine disorders has been developed. The validated method has successfully been tested using ERNDIM Diagnostic Proficiency Testing (DPT) samples and further clinical specimens from patients with various purine and pyrimidine disorders. Sample preparation is simple and assay duration is short, facilitating an easier inclusion of the assay into the diagnostic procedures.


Assuntos
Cromatografia Líquida/métodos , Erros Inatos do Metabolismo da Purina-Pirimidina/diagnóstico , Erros Inatos do Metabolismo da Purina-Pirimidina/urina , Espectrometria de Massas em Tandem/métodos , Adenina/análogos & derivados , Adenina/urina , Adolescente , Adulto , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/urina , Biomarcadores/urina , Criança , Pré-Escolar , Cromatografia Líquida/normas , Cromatografia Líquida/estatística & dados numéricos , Feminino , Humanos , Lactente , Masculino , Controle de Qualidade , Valores de Referência , Ribonucleotídeos/urina , Espectrometria de Massas em Tandem/normas , Espectrometria de Massas em Tandem/estatística & dados numéricos , Ureia/análogos & derivados , Ureia/urina , Uridina/análogos & derivados , Uridina/urina
19.
Molecules ; 24(3)2019 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-30678171

RESUMO

As an abundant post-transcriptional modification, dihydrouridine (D) has been found in transfer RNA (tRNA) from bacteria, eukaryotes, and archaea. Nonetheless, knowledge of the exact biochemical roles of dihydrouridine in mediating tRNA function is still limited. Accurate identification of the position of D sites is essential for understanding their functions. Therefore, it is desirable to develop novel methods to identify D sites. In this study, an ensemble classifier was proposed for the detection of D modification sites in the Saccharomyces cerevisiae transcriptome by using heterogeneous features. The jackknife test results demonstrate that the proposed predictor is promising for the identification of D modification sites. It is anticipated that the proposed method can be widely used for identifying D modification sites in tRNA.


Assuntos
RNA de Transferência/química , Saccharomyces cerevisiae/química , Máquina de Vetores de Suporte , Uridina/química , Algoritmos , Fenômenos Químicos , Conformação de Ácido Nucleico , Reprodutibilidade dos Testes , Uridina/análogos & derivados
20.
Methods ; 155: 88-103, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30529548

RESUMO

Many open questions in RNA biology relate to the kinetics of gene expression and the impact of RNA binding regulatory factors on processing or decay rates of particular transcripts. Steady state measurements of RNA abundance obtained from RNA-seq approaches are not able to separate the effects of transcription from those of RNA decay in the overall abundance of any given transcript, instead only giving information on the (presumed steady-state) abundances of transcripts. Through the combination of metabolic labeling and high-throughput sequencing, several groups have been able to measure both transcription rates and decay rates of the entire transcriptome of an organism in a single experiment. This review focuses on the methodology used to specifically measure RNA decay at a global level. By comparing and contrasting approaches and describing the experimental protocols in a modular manner, we intend to provide both experienced and new researchers to the field the ability to combine aspects of various protocols to fit the unique needs of biological questions not addressed by current methods.


Assuntos
Química Click/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Mensageiro/metabolismo , Coloração e Rotulagem/métodos , Transcriptoma , Animais , Biotina/análogos & derivados , Biotina/química , Linhagem Celular , Humanos , Estabilidade de RNA , RNA Mensageiro/genética , Tiouracila/análogos & derivados , Tiouracila/química , Tiouracila/metabolismo , Tiouridina/química , Tiouridina/metabolismo , Uracila/análogos & derivados , Uracila/química , Uracila/metabolismo , Uridina/análogos & derivados , Uridina/química , Uridina/metabolismo
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